Summary/Abstract Age-related macular degeneration (AMD) is the leading cause of vision loss in among elderly populations. Elevated homocysteine (Hcy), also known as hyperhomocysteinemia (HHcy) has been reported in patients with AMD; thereby suggesting an association between HHcy and the risk of AMD. Recently, we reported retinal changes similar to AMD in a mouse model of HHcy which lacks Cystathionine-?-synthase (cbs+/-) or received intravitreal injections of Hcy. These models showed significant retinal pigment epithelium (RPE) dysfunction and choroidal neovascularization (CNV) However, the lack of understanding the molecular/cellular mechanisms of these changes is a critical barrier in proposing Hcy as a therapeutic target in AMD. Our preliminary data show that HHcy-induced RPE dysfunction is associated with the upregulation of the N-methyl-D-aspartate (NMDAr) and GLUT1 receptors and increased glycolysis. Hence, we hypothesize that HHcy contributes to the pathogenesis of AMD via activation of the NMDAr and GLUT1 signaling pathways that induce the metabolic switch from oxidative phosphorylation to glycolysis. Therefore, elimination of excess Hcy through pharmacological or genetic intervention could be beneficial in the treatment of AMD. To test our hypothesis, we will conduct in vitro experiments, using RPE and choroidal endothelial cells (CEC) and in vivo using cbs+/-, wild type mice receiving intravitreal injection of Hcy and mice lacking the endothelial or RPE NMDAr (NMDAr-/-E or NMDAr-/-R respectively). Our specific aims include: 1: Testing the hypothesis that HHcy induces the metabolic switch from mitochondrial respiration to glycolysis via activation of GLUT1 in RPE cells: We will examine the changes in the retinal expression and localization of GLUT1, mitochondrial respiration, glycolysis and rate-limiting glycolytic enzymes in HHcy models. Moreover, we will determine the effect of GLUT1 inhibition on HHcy-induced RPE dysfunction and CNV. Aim 2: Testing the hypothesis that inhibition of NMDAr preserves RPE function and reduces the development of CNV under HHcy. We will examine the effects of pharmacological inhibition or genetic manipulation of the NMDAr on HHcy-induced RPE dysfunction and CNV. The effect of intravitreal injection of Hcy will be evaluated in NMDAr-/-E or NMDAr-/-R as compared to wild type and cbs+/- mice with or without NAMDAr inhibitors. Parallel in vitro experiments will be performed on RPE and CEC subjected to Hcy with or without NMDAr inhibitors followed by assessment of RPE function and angiogenic potential of CEC. Aim 3: Testing the hypothesis that elimination of excess Hcy by dietary supplementation or genetic/ pharmacological modifications prevents the progression of AMD. Hcy clearance will be enhanced in models of HHcy through two approaches, followed by assessment of RPE function and angiogenic potential of CEC: (a) Enhancing the remethylation pathway of Hcy metabolism using vitamins B6, B12 and folic acid supplementation. (b) Enhancing the transsulforation pathway of Hcy metabolism via CBS overexpression. Successful clearance of excess Hcy holds immense promise in the treatment of AMD.